The term “privileged structure” was first introduced by Evans et al. (Evans et al., J. Med. Chem. 31:2235 (1988)). The definition they provided for a privileged structure was “a single molecular framework able to provide ligands for diverse receptors.” This concept was partly suggested by the finding that variously derivatized benzodiazepines have been shown to be selective ligands for a variety of proteinaceous receptor surfaces. For example, Evans et al. discovered benzodiazepine analog antagonists of the peptide hormone cholecystokinin (CCK) with binding affinities in the same range as the natural peptide ligand CCK-8 for the peripheral CCK-A receptor (Evans et al., J. Med. Chem. 31:2235 (1988)). Subsequent developments in benzodiazepine and other privileged structures demonstrated that derivatives of these structures can provide potent and selective agonists and antagonists for additional members of the family of G-protein coupled receptors, even though the molecular size of these non-peptide ligands are much smaller than the natural peptide ligands that they mimic (Patchett et al., Annual Reports in Medicinal Chemistry, 35:289, Chap. 26 (2000)). Indeed, following the discovery of benzodiazepine privileged structures, a variety of additional privileged scaffolds (i.e. the substructures upon which the privileged structures are based) have been reported (Horton et al., Chemical Reviews 103(3):893 (2003)). Examples include biphenyl, diphenylmethane, benzopyran (Nicolaou et al., J. Am. Chem. Soc. 122:9939 (2000)), indole, and benzylpiperidine, among others (Horton et al., Chemical Reviews 103(3):893 (2003)). Focused combinatorial libraries based upon these privileged scaffolds have typically provided enriched “hit rates” of receptor agonists/antagonists, ion channels modulators, or enzyme inhibitors, when suitably decorated with appropriate side chains to impart both potency and selectivity.
The privileged structure concept suggests that the intersection of biological diversity space and chemical diversity space tends to occur in particular “nodes” and that privileged scaffolds are positioned within the chemical diversity space of these intersection nodes. This is the underlying concept for “gene-family” screening libraries (e.g. PCT International Publication No. WO 03/19183 to Grootenhuis et al.). From a molecular viewpoint, one can understand this concept in that nature, through the process of divergent evolution, tends to conserve protein molecular features that do not need to be changed and focuses on modifying particular features that are under evolutionary pressure to change in order to take on a new function (Murzin, Current Opinion in Structural Biology 8:380 (1998)). This is a much more efficient evolutionary process than attempting to de novo evolve a new protein for each new needed function. Consequently, one would expect that, within a evolutionarily related gene lineage, there will be certain general structural features within binding cavities that are conserved, whereas the finer details of the topology of these binding cavities will differ to reflect the particular specificities of each individual gene family member. The approximate size and shape of evolutionarily related binding cavities are the properties that would be expected to be conserved, whereas the particular amino acids lining the cavity and the exact position and size of resulting indentations or protrusions in the cavity are the features that would expect to be altered in individual proteins.
With this background in mind, one can envision how privileged scaffolds can be “designed” rather than relying on an empirical discovery, as has typically been the case in the past. The most efficient design approach would be to start with the X-ray or nuclear magnetic resonance (NMR) structure for a representative member of the evolutionarily related proteins. One can then utilize the range of molecular modeling technologies that are currently available to evaluate various candidate scaffolds for their ability to fit in the “middle” of the binding cavity and still allow space for the positioning of various appended side chains for favorable interactions with the amino acids that line the binding cavity. This central positioning of the privileged scaffold also provides a buffer zone around the scaffold, wherein the evolutionarily related proteins can display indentations and protrusions that can interdigitate with the side chains appended to the scaffold without interfering with the ability of the scaffold to bind in the cavity. The presence, nature, and positioning of the appended side chains on these scaffolds will then determine the specificity of the individual compounds due to their ability to interact with the amino acids forming the indentations and protrusions on the cavity surface. One can then synthesize probe libraries around each in silico identified privileged scaffold to determine experimentally which scaffolds are effective.
Designing screening libraries for identifying protein-protein interaction antagonists and, indeed, discovering protein-protein interaction antagonists in general have become a major challenge in drug discovery for the post-genomic era, due to the enormous number of potential untapped drug targets within this category. The ability of proteins to selectively bind to each other forms the foundation of much of biology, including disease biology. Cell architecture, signal transduction, and gene transcription are examples of important cell processes that are at least partly controlled by carefully scripted protein-protein interactions, and are processes that can lead to disease when abnormally altered. Many protein-protein “homodimers” bind very tightly through large, hydrophobic, surfaces, and their monomeric proteins are often unstable due to ready denaturization. In contrast, protein-protein “heterodimers” often bind less tightly, and their contact surfaces are typically more hydrophilic than those of homodimers. Consequently, these proteins tend to be stably folded as monomers and exist in equilibrium with their respective protein complexes. Protein-protein heterodimers are therefore generally considered more amenable targets for small molecule antagonists, because the equilibrium between free monomer and protein complex is more readily disrupted. This equilibrium can be altered by binding to one of the protein partners at the interaction interface, or through an allosteric site. Blocking a heterodimer complex that supports a particular disease biology may not necessarily require that the formation of heterodimer be completely inhibited; rather shifting the equilibrium may be sufficient. Discovering antagonists of protein-protein interactions is widely recognized as much more challenging than discovering enzyme inhibitors or receptor antagonists, even though these drug targets often involve protein substrates or ligands, respectively. The challenges and potential of this field, as well as progress to date, are summarized in several review articles, such as Gadek et al., Biochemical Pharmacology 65:1 (2003), Toogood, J. Med. Chem. 45(8):1543 (2002), and Cochran, Chemistry & Biology 7(4): R85 (2000). Gadek et al. pointed out that “ . . . as recently as 5 years ago the existence of small molecule antagonists was controversial. However, the antagonism of protein-protein interactions by small molecules is now well recognized, and the issue focuses on how these antagonists may be efficiently identified” (Gadek et al., Biochemical Pharmacology 65:1 (2003)).
Most protein-protein antagonists have been discovered by random screening rather than by design, and when the generally accepted MW 500 limit for compounds that are likely to be orally active drugs is applied to the reported antagonists, the majority have potencies in the 1-100 μM range (Boger et al., Angew. Chem. Int. Ed. 42:4138 (2003)). The results reported to date demonstrate that obtaining high potency, while maintaining a low molecular weight, is particularly challenging for these drug targets as compared to the more traditional drug targets. The maximum binding affinity possible for a small molecule to a particular protein-protein interaction target will depend upon the nature of the binding interface of the two proteins. Although protein-protein interactions typically involve a large surface area of contact, i.e. greater than 600 Å2, most of the binding affinity is derived from “hot spots” located near the center of the contact surface (Bogan et al., J. Mol. Biol. 280:1 (1998)). In many cases, these hot spots are not deep cavities in the protein surface, in contrast to the deep crevice binding cavities typical for enzyme inhibitors/substrates. A detailed analysis of protein-protein interactions surfaces indicated that the binding affinity attributed to these hot spots is increased by the exclusion of water from this region of the interface by a surrounding hydrophobic “O-ring” of residues (Bogan et al., J. Mol. Biol. 280:1 (1998)). The exclusion of water by the hydrophobic O-ring was proposed to strengthen the interactions in the hot spots by decreasing the dielectric of the microenvironment, thereby strengthening polar interactions, and by reducing the rate of dissociation between hydrophobic groups. Consequently, binding affinity for small molecule protein-protein antagonists may be derived from two factors: 1) the shape and electronic surface complementarity of the small molecule for the hot spot, and 2) the ability of the small molecule to also shield key interactions from bulk solvent. The deeper the binding cavity forming a hot spot the more traditional binding affinity factors will be involved. The more open the binding cavity the more solvent shielding may be important.
The factors described above result in a much higher level of challenge for rationally designing tight binding, low molecular weight, antagonists of protein-protein interactions, as compared to the traditional drug targets.
Experience to date has shown that random high-throughput screening for antagonists of protein-protein interactions has generally been much less successful than has similar screening for other drug targets such as receptor antagonists, enzyme inhibitors or ion channel blockers. This may be due to using screening libraries not suitably biased towards small molecules that have the needed topology for binding to the target protein surface. In order to appreciate the scale of the problem, one needs to recognize that the volume of oral drug-sized chemical diversity space (i.e. MW ca. 500 or less) is enormous. Estimates have put the number of possible compounds with molecular weights of 500 Da or less at 10200 compounds. Even when the typical filters for selecting only drug-sized molecules that are also expected to have drug-like properties (beyond MW) are applied, this number only reduces to 1060 compounds (see Horton et al., Chemical Reviews 103(3):893 (2003)). As Horton et al. points out, “the proportion of these drug-like molecules synthesized to date has been estimated as one part in 1057, or roughly the ratio of the mass of one proton to the mass of the sun” (Horton et al., Chemical Reviews 103(3):893 (2003)). Clearly, there is a pressing need to develop better methods for “targeting” the region of chemical diversity space wherein drug-like antagonists of “drugable” protein-protein interactions reside. It is impossible to cover the total volume of drug-like chemical diversity space with any reasonable density by random broad screening. Toogood, J. Med. Chem. 45(8):1543 (2002) states that “currently, there are no general techniques or approaches that will reliably illuminate the path toward the synthesis of potent and effective drug-like protein-protein binding inhibitors.” Toogood then goes on to state “it is hoped that over time some themes may emerge, highlighting for example particular generic structures that may form a basis for protein-protein binding inhibitors. Compound libraries then can be populated with compounds exemplifying these structures, increasing the chances of lead discovery through high throughput screening. Perhaps then the trepidation that protein-protein binding currently imbues in many medicinal chemists will be overcome, and the rich opportunities available for drug discovery finally will be recognized” (Toogood, J. Med. Chem. 45(8):1543 (2002)).
Transcription factor complexes are responsible for much of the regulation of gene expression (Wolgerger, Ann. Rev. Biophys. Biomol. Struct. 28:29 (1999)) and therefore offer many potential protein-protein interaction drug targets (Emery et al., Trends in Pharmacological Sciences, 22:233 (2001)), including anti-cancer targets (Karamouzis et al., Clinical Cancer Research 8:949 (2002)). The 39 members of the HOX family (Scott, Cell. 71:551 (1992)) and 4 members of the PBX family (Knoepfler et al., Mech. Dev. 63:5 (1997)) are proteins that bind DNA as heterodimers to form transcription factor complexes. The numerous heterodimeric HOX/PBX combinations play critical and complex roles in transcriptional regulation during patterning in early embryonic development (Knoepfler et al., Mech. Dev. 63:5 (1997)) and many are utilized again in specific tissues of the adult (van Oostveen et al., Leukemia 13:1675 (1999)). A variety of cancers display altered HOX gene regulation (Maroulakou et al., Anticancer Research 23:2101 (2003)). For example, in leukemias and lung cancers, HOX genes are overexpressed and consequently antagonists of the HOX/PBX complexes controlling these genes offers the potential for novel anticancer drugs. Also, small molecule antagonists of individual (or subsets) HOX/PBX complexes can be used as pharmacological tools to investigation their function.
It would be advantageous to find small molecule antagonists of protein-protein interactions, such as HOX-PBX protein-protein interactions.
The present invention is directed to achieving this objective.